This project aims to define the dynamic molecular mechanism by which protein synthesis - translation - is initiated in eukaryotes. Regulated translation is fundamental to the function of the cell; proteins must be synthesized with spatial and temporal precision to ensure cellular viability. In contrast, misregulated translation has dire consequences for human health and is central to many diseases including cancer, viral infection, developmental defects, and autism. Initiation of translation is its most regulated phase and is a complex process involving the ribosomal subunits, mRNA, and at least 23 polypeptides that guide the formation of an elongation-competent 80S ribosomal particle. In the K99 phase, existing methodologies to study early translation initiation will be developed in a mentored setting that expands Dr. O'Leary's abilities to implement single-molecule techniques and develops skills needed to work with eukaryotic cells. We will use single- molecule fluorescence microscopy to determine the dynamics - the time-evolution of biomolecular composition and conformation - that underpin key phases of the initiation process. The proposed research builds on the single-molecule platform Dr. O'Leary has developed to study the earliest part of initiation - recognition of the mRNA 5' cap by the Saccharomyces cerevisiae cap-binding protein eIF4E and the modulation of this process by other components of the pre-initiation complex. We will expand this technology to uncover the mechanism of ribosomal scanning, the process through which the mRNA start codon is located. We will develop an assay for the rate of scanning and use this to determine the effects of the mRNA 5' untranslated region and initiation factors on the scanning process (Aim 1). We will define the role of mRNA-protein interactions in coordinating the scanning process specifically, and the dynamics of initiation more generally (Aim 2). These K99-phase studies will be carried out using S. cerevisiae translation components as a model system. While yeast is an invaluable model for establishing the fundamentals of the initiation mechanism, there are differences between the yeast and human translation machinery that must be taken into account when applying knowledge obtained with yeast to human translation. In the R00 phase, we will address these differences by using the skills developed during the mentored phase and the knowledge resulting from Aims 1 and 2. To this end, in the R00 phase we will reconstitute the human translation initiation machinery and characterize key mechanistic differences (Aim 3). The combined results from Aims 1 - 3 will provide the mechanistic understanding needed to interrogate important regulatory mechanisms central to human health (Aim 4). In particular, we will examine the roles of translational control by microRNAs and mRNA degradation. The combination of mentored support, skills, and data obtained in the K99 phase will provide Dr. O'Leary a springboard to achieving independence as a researcher in the R00 phase and beyond. The results of our studies will provide new insights into fundamental aspects of cellular function, and will define new paradigms relevant to biology and medicine.